Worldwide, diarrhea claims several million lives annually, mostly those of infants. Poverty, crowding, and contaminated water supplies all contribute. Although its incidence is much lower in the more affluent nations, diarrhea remains one of the two most common visits to pediatric emergency rooms and is also common among the institutionalized elderly. Diarrhea is also a troublesome gastrointestinal complication of diabetes. Major salt and water absorption in the intestine is mediated by electroneutral NaCl absorption, consisting of a Na+/H+ exchanger coupled with a Cl-/HCO3- exchanger. The Na+/H+ exchanger isoform 3 (NHE3) is often targeted in many diarrheal diseases. However, the molecular basis and biological factors regulating NHE3 are not fully understood. This is an important aspect as a better understanding of the mechanisms activating NHE3 should aid the development of improved therapeutic modalities to combat diarrhea caused by the inhibition of Na+/H+ exchange. During the previous funding cycle, we have demonstrated that lysophosphatidic acid (LPA) activates NHE3 via LPA5R in colon epithelial cells and showed that LPA is a potent activator of NHE3 and fluid absorption in mouse intestine. In addition, we showed that the effect of LPA involves apical epidermal growth factor receptor (EGFR). However, the mechanism of LPA-induced EGFR transactivation and the activation of NHE3 are incompletely studied. We aim to determine the molecular mechanism of NHE3 regulation by LPA and test the importance of LPA5R in vivo (Specific Aim 1). Diabetic diarrhea is a troublesome gastrointestinal complication of type 1 diabetes. Our preliminary data show that mice with diabetes induced by streptozotocin (STZ) develop diarrhea and NHE3 activity is significantly attenuated. Our study further shows that insulin stimulates NHE3 activity in STZ-treated mice, although the underlying mechanisms are unknown. We will determine the molecular mechanism of NHE3 activation by insulin and test the utility of LPA as an activator of NHE3 and fluid absorption in the type 1 diabetic model in rodent (Specific Aim 2). Our preliminary data show that metformin commonly used for glycemic control in type 2 diabetes inhibits NHE3 activity, further suggesting a causal role of NHE3 in diabetes-related diarrhea. Our data show that NHE3 is ubiquitinated in response inhibitory agents, such as metformin, phorbol ester, and forskolin. Surprisingly, we observed a significant difference in ubiquitination of NHE3 between human and non-human. The status of NHE3 ubiquitination and its role have never been investigated. In Specific Aim 3, we will investigate the mechanisms that regulate NHE3 ubiquitination and the importance of ubiquitination in NHE3 regulation and EPEC-induced diarrhea. Successful completion of the proposed work will increase the knowledge of molecular mechanism of NHE3 regulation and will provide insights into improved strategies for treatment and prevention of diarrheal diseases.